NR4A2 is regulated by gastrin and influences cellular responses of gastric adenocarcinoma cells

Abstract

The peptide hormone gastrin is known to play a role in differentiation, growth and apoptosis of cells in the gastric mucosa. In this study we demonstrate that gastrin induces Nuclear Receptor 4A2 (NR4A2) expression in the adenocarcinoma cell lines AR42J and AGS-GR, which both possess the gastrin/CCK2 receptor. In vivo, NR4A2 is strongly expressed in the gastrin responsive neuroendocrine ECL cells in normal mucosa, whereas gastric adenocarcinoma tissue reveals a more diffuse and variable expression in tumor cells. We show that NR4A2 is a primary early transient gastrin induced gene in adenocarcinoma cell lines, and that NR4A2 expression is negatively regulated by inducible cAMP early repressor (ICER) and zinc finger protein 36, C3H1 type-like 1 (Zfp36l1), suggesting that these gastrin regulated proteins exert a negative feedback control of NR4A2 activated responses. FRAP analyses indicate that gastrin also modifies the nucleus-cytosol shuttling of NR4A2, with more NR4A2 localized to cytoplasm upon gastrin treatment. Knock-down experiments with siRNA targeting NR4A2 increase migration of gastrin treated adenocarcinoma AGS-GR cells, while ectopically expressed NR4A2 increases apoptosis and hampers gastrin induced invasion, indicating a tumor suppressor function of NR4A2. Collectively, our results uncover a role of NR4A2 in gastric adenocarcinoma cells, and suggest that both the level and the localization of NR4A2 protein are of importance regarding the cellular responses of these cells.

Figure 1. NR4A2 is induced by gastrin.

A: Temporal profiles of gastrin induced NR4A2 mRNA expression in pancreatic adenocarcinoma cells (AR42J). The panels show data from three independent microarray time series experiments (accession numbers E-MATAB-1268 and GSE32869); and the data points are presented as normalized log₂-transformed signal intensities. Experiment 1: mRNA expression level for untreated (green line) and sustained gastrin treated (blue line) cells. Experiment 2: mRNA level in cells treated in a sustained mode (14 h of continuous presence of gastrin) and in a transient mode (gastrin was removed after 1 h of treatment). Experiment 3: sustained gastrin treatment was measured in the presence (orange line) and absence (blue line) of cycloheximide (CHX) at 6 different time points between 1 and 10 h. Green and grey lines show mRNA levels in untreated and CHX treated control cells, respectively. All data points are mean of two biological replicates. Gastrin (10 nM) treated and untreated control cells were grown in parallel and harvested (pool of 2-3 technical replicates) at several time points, as indicated in the panels. In experiments with transient versus sustained gastrin treatment, the growth medium of untreated and gastrin treated cells was removed 1 h after gastrin treatment; the cells were then washed with serum-free medium before fresh serum-free medium with gastrin (sustained gastrin treated cells) or without gastrin (transiently gastrin treated or untreated cells) was added. In experiments with the protein synthesis inhibitor cycloheximide (CHX), pre-treatments with CHX (10 µg/ml) were initiated 30 min before gastrin (10 nM) was added. B: NR4A2 mRNA and protein level in gastrin treated (5 nM) AGS-G_R cells. qRT-PCR data shown are mean ± SEM of four biological replicas. Western blot image shows NR4A2 protein. Immunostaining for NR4A2 in normal gastric oxyntic mucosa is shown in panels C-E: Strong NR4A2 immunoreactivity (C) in scattered single cells in normal gastric oxyntic mucosa. Overlap between the cells showing strong NR4A2 immunoreactivity (D) and CgA immunoreactive neuroendocrine cells (E) in serial sections (C at x400 magnification, E and F at x1000 magnification).

Figure 2. NR4A2 activates NBRE promoter elements.

A: Gastrin-induced NBRE-luc activation. Data represent one of two biological replicas. B: The effect of NR4A2 siRNA on gastrin-induced NBRE activation. Data represent mean ± SEM of four biological replicas (** p<0.01, * p=0.1). C-D: Effect of specific inhibitors of PKA (H-89, 10µM), PI3K (LY 294002, 10µM) or PKC (GF 109203x, 3.5µM) on (C) gastrin-induced NR4A2 gene expression and (D) gastrin-induced NBRE activation. Data represent one of three biological replicas; mean ± SD of six technical replicas.

Figure 3. Negative regulation of gastrin-induced NR4A2 expression.

A: AGS-G_R cells transfected with NR4A2-luc and ICER expression plasmids or empty vector. Cells were treated with gastrin for 6 h prior to measurement of NR4A2 activity. Data shown represent mean ± SEM of five biological replicas (** p<0.03, * p = 0.06). B: AGS-G_R cells transfected with NBRE-luc and ICER expression plasmid or empty vector and treated with gastrin for 4 h prior to measurement of NBRE activity. Data shown represent mean ± SEM of four biological replicas (** p<0.03). C: AGS-G_R cells were transfected with pZfp36l1 expression plasmid or empty vector and treated with gastrin (5 nM) NR4A2 mRNA expression was measured by qRT-PCR. Data shown represent one of three biological replicas; mean ± SD of three technical replicas is shown. D: Cyclin L1 represents one of three control genes examined.

Figure 4. Gastrin treatment influences nucleus-cytosolic shuttling of NR4A2.

A: Intracellular localization of NR4A2 protein in response to gastrin treatment. AGS-G_R cells transfected with pNR4A2-EGFP. B: Images of gastrin treated (10 nM) AGS-G_R cells expressing pNR4A2-EGFP before, during and after bleaching of a nucleus area for 7.5 sec. The circle indicates the area of the bleach spot. C: Normalized FRAP curve for the cytosol of gastrin treated AGS-G_R cells. D: Diffusion time in untreated and gastrin treated nucleus and cytosol. E: To determine cell viability, cells were transfected with pNR4A2-EGFP or a control plasmid (pH3.1-EGFP). After 48 h AGS-G_R cells were detached by Accutase treatment, labeled with annexin V Alexa Fluor 647 and analyzed by flow cytometry. Annexin-V positive cells were considered as apoptotic. Results are shown as % apoptotic cells of the total number of counted EGFP positive or EGFP negative cells. Data are representative of three biological replicas; mean ± SD of three technical replicas is shown.

Figure 5. NR4A2 suppresses gastrin-induced migration and invasion.

A: Real-time cell migration monitored (0-24 h) in AGS-G_R cells transfected with siNR4A2 or siCtr, with or without gastrin treatment (10 nM). Results show one representative of three biological replicas; mean ±SD of three technical replicas. B: Invasion assay with AGS-G_R cells transfected with pCMX-NR4A2 or pCMX (control) was performed in 24-well plates containing 8-µm pore Matrigel-coated inserts (with or without 0.3 nM gastrin). Cells invading the lower surface of the membrane were stained with Reastain Quick-Diff reagents and total numbers of cells in 5 fields per membrane were counted. The mean of three independent experiments is shown.

Figure 6. Immunostaining of NR4A2 in gastric adenocarcinoma.

A-B: NR4A2 immunoreactivity in normal oxyntic mucosa showing strong intensity in scattered single cells (neuroendocrine cells) and weaker staining intensity in the other epithelial cells. C-F: NR4A2 immunoreactivity in gastric adenocarcinomas of intestinal (C-D) and diffuse (E-F) type, showing a general staining in tumor cells with mixed nuclear or cytoplasmic localization and variable intensities. (A, C, E at x200 magnification, with boxes representing B, D and F at x400 magnification).